Characterization of mechanism of action of hydrogen sulfide (H2S) in the regulation of smooth muscle function

Hydrogen sulfide (H2S) is receiving increasing interest, as much as nitric oxide (NO) and carbon monoxide have received previously, to understand its physiological functions as it meets all the criteria to define as a third gasotransmitter. Endogenous synthesis from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) and the function of H2S as an inhibitor of smooth muscle contraction in gastrointestinal tract are known. However, the loci of generation and action of H2S, and the mechanism of inhibition of contraction are unknown. Hence, my aims in the present study are to: i) identify the expression of enzymes in smooth muscle, ii) determine the effects of endogenously released and exogenously applied H2S on smooth muscle function; and iii) identify the targets and mechanism involved in mediating the effects of H2S using isolated smooth muscle cells from rabbit colon. I have identified the expression of CSE, but not CBS, in smooth muscle and demonstrated that L-cysteine (an activator of CSE) and NaHS (H2S donor): 1) inhibited carbachol-induced contraction in muscle strips and isolated muscle cells that was independent of KATP channels, a known S-sulfhydration target of H2S; 2) induced S-sulfhydration of small G protein, RhoA leading to inhibition of RhoA and Rho kinase activities, a key pathway in the sustained smooth muscle contraction; and 3) inhibited PDE5 activity leading to augmentation NO-induced cGMP formation and muscle relaxation. Sodium nitroprusside (an NO donor) induced an increase in H2S production via PKG-dependent phosphorylation and activation of CSE. We conclude that smooth muscle cells selectively express CSE, and endogenous generation of H2S via activation of CSE inhibits muscle contraction and augments muscle relaxation. Inhibition of contraction is mediated via S-sulfhydration of RhoA and suppression of RhoA/Rho kinase pathway. Augmentation of relaxation is mediated via inhibition of PDE5 activity and stimulation of cGMP/PKG pathway, which in addition initiates generation of H2S via PKG-mediated phosphorylation and activation of CSE. The findings are important in providing the underlying mechanisms involved in the regulation of smooth muscle function by H2S and could offer insights for the development of therapeutic agents that may act on smooth muscle in the gut to treat motility disorders.